Bortezomib Treatment can Overcome Glucocorticoid Resistance in Childhood B-cell Precursor Acute Lymphoblastic Leukemia Cell Lines.

BACKGROUND: The response to initial glucocorticoid (gc) treatment is a reliable stratification factor in childhood acute lymphoblastic leukemia (ALL) and may predict the response to multi-agent chemotherapy. In a former study we detected that the valosin-containing protein (VCP, cdc48), a member of the ubiquitin proteasome degradation system (UPS), is altered in gc-resistant leukemic cells suggesting that the associated pathways might be involved in chemotherapy resistance in childhood ALL. METHODS: Human B-cell precursor leukemia cell lines, gc-resistant MHH-cALL-2 and gc-sensitive MHH-cALL-3, were treated with prednisolone and various concentrations of bortezomib. Viability and apoptosis rates were determined. RESULTS: Both cell lines showed a dose-dependent increase in caspase activity after bortezomib single treatment. The gc-sensitive cells showed an additive effect after combined treatment with prednisolone and bortezomib. In contrast, both cell lines showed a reduced viability and enhanced propidium iodide positivity after combined treatment as determined by flow cytometry. Western blot analyses of poly-(ADP-ribose) polymerase 1 (PARP-1) suggested that combined treatment promote necrotic cleavage of PARP-1 in gc-resistant cells. Furthermore, after prednisolone treatment the UPS associated proteins VCP and NFκB-inhibitor IκBα were differentially modulated in gc-resistant cells. CONCLUSIONS: The proteasome inhibitor bortezomib seems to sensitize gc-resistant childhood ALL cells for prednisolone-induced cell death.

Minimal residual disease-based treatment is adequate for relapse-prone childhood acute lymphoblastic leukemia with an intrachromosomal amplification of chromosome 21: the experience of the ALL-BFM 2000 trial.

BACKGROUND: Recently, the UK CCLG and COG reported that an intrachromosomal amplification of chromosome 21 (iAMP21) in acute lymphoblastic leukemia (ALL) loses its adverse prognostic impact with intensified therapy. PATIENT AND METHODS: We evaluated the prognosis of iAMP21 among patients from the ALL-BFM (Berlin-Frankfurt-Münster) 2000 trial with 46 of 2 637 (2%) patients iAMP21+. RESULTS: 8-year event-free-survival (EFS, 64 ± 8% vs. 81 ± 1%, p=0.0026) and cumulative incidence of relapse (CIR, 29 ± 8% vs. 14 ± 1%, p=0.008) of the iAMP21 cases were significantly worse compared with non-iAMP21 patients. Within the MRD low-risk group, iAMP21 cases (n=14) had an inferior 8-year EFS (76 ± 12% vs. 92 ± 1%, p=0.0081), but no increased CIR (10 ± 10% vs. 6 ± 1%, p=0.624). Within the MRD intermediate-risk group, iAMP21 cases (n=27) had a worse 8-year EFS (56 ± 11% vs. 78 ± 2%, p=0.0077) and CIR (44 ± 11% vs. 20 ± 2%, p=0.003) with 6/10 relapses occurring after 2 years. CONCLUSIONS: Conclusively, we believe that there is no necessity for enrolling all iAMP21 patients into the high-risk arm of ongoing ALL-BFM trials because MRD low-risk patients have a moderate relapse risk under current therapy. Whether the increased relapse risk in MRD intermediate-risk patients can be avoided by late treatment intensification remains to be answered by the AIEOP-BFM ALL 2009 trial randomly using protracted pegylated L-asparaginase during delayed intensification and early maintenance.

Prediction of outcome by early response in childhood acute lymphoblastic leukemia.

BACKGROUND: In the ALL-BFM studies for treatment of acute lymphoblastic leukemia, reduction of leukemic blasts in peripheral blood after a one-week prednisone pre-phase - the so-called prednisone response - has been used for risk stratification since the 1980s and has been one of the most relevant factors for identification of high-risk patients. In the trial ALL-BFM 95, early cytomorphological marrow response on day 15 of induction therapy was prospectively evaluated and its prognostic value was analyzed in comparison to the prednisone response and other established prognostic factors. RESULTS: Compared to prednisone response, day 15 marrow response was superior in outcome prediction - yet with differential effect depending on blast lineage. Outcome was poor in T cell leukemia patients with prednisone poor-response independent of day 15 marrow response, whereas among patients with prednisone good-response different risk groups could be identified by day 15 marrow response. In contrast, prednisone response lost prognostic significance in precursor B cell leukemia when stratified by day 15 marrow response. CONCLUSIONS: Selective addition of day 15 marrow response to conventional stratification criteria applied on ALL-BFM 95 may significantly improve risk-adapted treatment delivery. Even though cutting-edge trial risk stratification is meanwhile dominated by minimal residual disease evaluation, an improved conventional risk assessment, as presented here, could be of great importance to countries lacking the technical and/or financial resources associated with the application of minimal residual disease analysis.

Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Folate metabolic gene polymorphisms and childhood acute lymphoblastic leukemia: a case-control study.

We genotyped six folate metabolic pathway genes for 11 polymorphisms in 460 cases of childhood acute lymphoblastic leukemia (ALL) and 552 ethnically matched controls. None of the polymorphisms except the 66A>G (I22M) in the 5-methyltetrahydrofolate-homocysteine methyltransferase reductase (MTRR) gene showed any effect on disease risk. The carriers of the G-allele were associated with a marginal decreased risk of ALL (gender-adjusted global P=0.03; multiple-testing corrected P=0.25). Analysis of four polymorphisms in the MTRR gene showed statistically significant differences in haplotype distribution between cases and controls (global P<0.0001). The haplotypes GCAC (odds ratio (OR) 0.5, 95% confidence interval (CI) 0.4-0.6) and ATAC (OR 0.5, 95% CI 0.3-0.6) were associated with a reduced risk and the haplotypes ACAC (OR 2.3, 95% CI 1.8-2.9) and GTAC (OR 1.8, 95% CI 1.4-2.3) with an increased risk. The genotype-combination analyses indicated that the best model stratifies cases and controls based on the 66A>G and the 524C>T polymorphisms in the MTRR gene (global P=0.03). Our results suggest that, besides a weak association of childhood ALL with the 66A>G polymorphism, haplotypes within the MTRR gene may, in part, account for population-based differences in risk.

NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

Mixed-lineage leukemia (MLL)-rearranged (MLLr) infant B-cell acute lymphoblastic leukemia (iMLLr-B-ALL) has a dismal prognosis and is associated with a pro-B/mixed phenotype, therapy refractoriness and frequent central nervous system (CNS) disease/relapse. Neuron-glial antigen 2 (NG2) is specifically expressed in MLLr leukemias and is used in leukemia immunophenotyping because of its predictive value for MLLr acute leukemias. NG2 is involved in melanoma metastasis and brain development; however, its role in MLL-mediated leukemogenesis remains elusive. Here we evaluated whether NG2 distinguishes leukemia-initiating/propagating cells (L-ICs) and/or CNS-infiltrating cells (CNS-ICs) in iMLLr-B-ALL. Clinical data from the Interfant cohort of iMLLr-B-ALL demonstrated that high NG2 expression associates with lower event-free survival, higher number of circulating blasts and more frequent CNS disease/relapse. Serial xenotransplantation of primary MLL-AF4+ leukemias indicated that NG2 is a malleable marker that does not enrich for L-IC or CNS-IC in iMLLr-B-All. However, NG2 expression was highly upregulated in blasts infiltrating extramedullar hematopoietic sites and CNS, and specific blockage of NG2 resulted in almost complete loss of engraftment. Indeed, gene expression profiling of primary blasts and primografts revealed a migratory signature of NG2+ blasts. This study provides new insights on the biology of NG2 in iMLLr-B-ALL and suggests NG2 as a potential therapeutic target to reduce the risk of CNS disease/relapse and to provide safer CNS-directed therapies for iMLLr-B-ALL.

Correction: NG2 antigen is involved in leukemia invasiveness and central nervous system infiltration in MLL-rearranged infant B-ALL.

The original version of this Article contained an error in the spelling of the author Juan Carlos Rodriguez-Manzaneque, which was incorrectly given as J Carlos Rodríguez-Manzaneque. This has now been corrected in both the PDF and HTML versions of the Article.

DNA cleavage within the MLL breakpoint cluster region is a specific event which occurs as part of higher-order chromatin fragmentation during the initial stages of apoptosis.

A distinct population of therapy-related acute myeloid leukemia (t-AML) is strongly associated with prior administration of topoisomerase II (topo II) inhibitors. These t-AMLs display distinct cytogenetic alterations, most often disrupting the MLL gene on chromosome 11q23 within a breakpoint cluster region (bcr) of 8.3 kb. We recently identified a unique site within the MLL bcr that is highly susceptible to DNA double-strand cleavage by classic topo II inhibitors (e.g., etoposide and doxorubicin). Here, we report that site-specific cleavage within the MLL bcr can be induced by either catalytic topo II inhibitors, genotoxic chemotherapeutic agents which do not target topo II, or nongenotoxic stimuli of apoptotic cell death, suggesting that this site-specific cleavage is part of a generalized cellular response to an apoptotic stimulus. We also show that site-specific cleavage within the MLL bcr can be linked to the higher-order chromatin fragmentation that occurs during the initial stages of apoptosis, possibly through cleavage of DNA loops at their anchorage sites to the nuclear matrix. In addition, we show that site-specific cleavage is conserved between species, as specific DNA cleavage can also be demonstrated within the murine MLL locus. Lastly, site-specific cleavage during apoptosis can also be identified at the AML1 locus, a locus which is also frequently involved in chromosomal rearrangements present in t-AML patients. In conclusion, these results suggest the potential involvement of higher-order chromatin fragmentation which occurs as a part of a generalized apoptotic response in a mechanism leading to chromosomal translocation of the MLL and AML1 genes and subsequent t-AML.

Consensus guidelines for microarray gene expression analyses in leukemia from three European leukemia networks.

A plethora of studies have documented that gene expression profiling using DNA microarrays for various types of hematological malignancies provides novel information, which may have diagnostic and prognostic implications. However, to successfully use microarrays for this purpose, the quality and reproducibility of the whole procedure need to be guaranteed. Critical steps of the method are handling, processing and storage of the leukemic sample, purification of tumor cells (or lack thereof), RNA extraction methods, quality control of RNA, labeling techniques, hybridization, washing, scanning, spot filtering, normalization and initial interpretation, and finally the biostatistical analysis. These items have been extensively discussed and evaluated in different multi-center quality rounds within the three networks, that is, I-BFM-SG, the German Competence Network 'Acute and Chronic Leukemias' and the European LeukemiaNet. Based on the exchange of knowledge and experience between the three networks over the last few years, we have formulated guidelines for performing microarray experiments in leukemia. We confine ourselves to leukemias, but many of these requirements also apply to lymphomas or other clinical samples, including solid tumors.

The MLL recombinome of acute leukemias.

Chromosomal rearrangements of the human MLL gene are a hallmark for aggressive (high-risk) pediatric, adult and therapy-associated acute leukemias. These patients need to be identified in order to subject these patients to appropriate therapy regimen. A recently developed long-distance inverse PCR method was applied to genomic DNA isolated from individual acute leukemia patients in order to identify chromosomal rearrangements of the human MLL gene. We present data of the molecular characterization of 414 samples obtained from 272 pediatric and 142 adult leukemia patients. The precise localization of genomic breakpoints within the MLL gene and the involved translocation partner genes (TPGs) was determined and several new TPGs were identified. The combined data of our study and published data revealed a total of 87 different MLL rearrangements of which 51 TPGs are now characterized at the molecular level. Interestingly, the four most frequently found TPGs (AF4, AF9, ENL and AF10) encode nuclear proteins that are part of a protein network involved in histone H3K79 methylation. Thus, translocations of the MLL gene, by itself coding for a histone H3K4 methyltransferase, are presumably not randomly chosen, rather functionally selected.

Polymorphic variation in TPMT is the principal determinant of TPMT phenotype: A meta-analysis of three genome-wide association studies.

Thiopurine-related hematotoxicity in pediatric acute lymphoblastic leukemia (ALL) and inflammatory bowel diseases has been linked to genetically defined variability in thiopurine S-methyltransferase (TPMT) activity. While gene testing of TPMT is being clinically implemented, it is unclear if additional genetic variation influences TPMT activity with consequences for thiopurine-related toxicity. To examine this possibility, we performed a genome-wide association study (GWAS) of red blood cell TPMT activity in 844 Estonian individuals and 245 pediatric ALL cases. Additionally, we correlated genome-wide genotypes to human hepatic TPMT activity in 123 samples. Only genetic variants mapping to chromosome 6, including the TPMT gene region, were significantly associated with TPMT activity (P < 5.0 × 10-8 ) in each of the three GWAS and a joint meta-analysis of 1,212 cases (top hit P = 1.2 × 10-72 ). This finding is consistent with TPMT genotype being the primary determinant of TPMT activity, reinforcing the rationale for genetic testing of TPMT alleles in routine clinical practice to individualize mercaptopurine dosage.

Identification of a genetically defined ultra-high-risk group in relapsed pediatric T-lymphoblastic leukemia.

In the search for genes that define critical steps of relapse in pediatric T-cell acute lymphoblastic leukemia (T-ALL) and can serve as prognostic markers, we performed targeted sequencing of 313 leukemia-related genes in 214 patients: 67 samples collected at the time of relapse and 147 at initial diagnosis. As relapse-specific genetic events, we identified activating mutations in NT5C2 (P=0.0001, Fisher's exact test), inactivation of TP53 (P=0.0007, Fisher's exact test) and duplication of chr17:q11.2-24.3 (P=0.0068, Fisher's exact test) in 32/67 of T-ALL relapse samples. Alterations of TP53 were frequently homozygous events, which significantly correlated with higher rates of copy number alterations in other genes compared with wild-type TP53 (P=0.0004, Mann-Whitney's test). We subsequently focused on mutations with prognostic impact and identified genes governing DNA integrity (TP53, n=8; USP7, n=4; MSH6, n=4), having key roles in the RAS signaling pathway (KRAS, NRAS, n=8), as well as IL7R (n=4) and CNOT3 (n=4) to be exclusively mutated in fatal relapses. These markers recognize 24/49 patients with a second event. In 17 of these patients with mostly refractory relapse and dire need for efficient treatment, we identified candidate targets for personalized therapy with p53 reactivating compounds, MEK inhibitors or JAK/STAT-inhibitors that may be incorporated in future treatment strategies.

A genome-wide association study identifies risk loci for childhood acute lymphoblastic leukemia at 10q26.13 and 12q23.1.

Genome-wide association studies (GWASs) have shown that common genetic variation contributes to the heritable risk of childhood acute lymphoblastic leukemia (ALL). To identify new susceptibility loci for the largest subtype of ALL, B-cell precursor ALL (BCP-ALL), we conducted a meta-analysis of two GWASs with imputation using 1000 Genomes and UK10K Project data as reference (totaling 1658 cases and 7224 controls). After genotyping an additional 2525 cases and 3575 controls, we identify new susceptibility loci for BCP-ALL mapping to 10q26.13 (rs35837782, LHPP, P=1.38 × 10-11) and 12q23.1 (rs4762284, ELK3, P=8.41 × 10-9). We also provide confirmatory evidence for the existence of independent risk loci at 9p21.3, but show that the association marked by rs77728904 can be accounted for by linkage disequilibrium with the rare high-impact CDKN2A p.Ala148Thr variant rs3731249. Our data provide further insights into genetic susceptibility to ALL and its biology.

Immunosurveillance of childhood ALL: polymorphic interferon-gamma alleles are associated with age at diagnosis and clinical risk groups.

Interferon-gamma (IFN-gamma) has been implicated as an important mediator of antitumor immunity in murine model systems. To determine whether a CA-repeat associated with differential NFkappaB-binding and IFN-gamma-expression levels may influence the incidence, manifestation and early clinical treatment response of childhood acute lymphoblastic leukemia, we performed PCR-based genotyping of 393 patients with ALL and 207 healthy controls. We could not find any differences in the allele distribution comparing patients and controls. However, when we further analyzed the allele frequencies with respect to age of clinical manifestation, we found that patients with B-lineage ALL showing the IFN-gamma high-expressing genotype presented at a more advanced age compared to those patients with intermediate and low-expressing genotypes (median 6 vs 4.4 years, P=0.01). Furthermore, we found a significantly higher number of low expressors in the group of high-risk patients (HR n=32 and MR/SR n=266, P=0.025, defined by prednisone response, cytological remission and minimal residual disease (MRD)) with B-lineage ALL. Thus, we provide evidence that polymorphic IFN-gamma alleles are associated with age at clinical presentation and risk groups such as prednisone response in B-lineage ALL, suggesting distinct effects of IFN-gamma in immunosurveillance and early response to steroid therapy.

Preexisting conditions in pediatric ALL patients: Spectrum, frequency and clinical impact.

INTRODUCTION: The etiology of acute lymphoblastic leukemia remains undisclosed in the majority of cases. A number of rare syndromic conditions are known to predispose to different forms of childhood cancer including ALL. The present study characterized the spectrum and clinical impact of preexisting diseases in a cohort of ALL patients from Germany, Austria and Switzerland with a focus on genetic diseases predisposing to cancer development. METHODS: Retrospective database and study chart review included all patients from Germany, Austria and Switzerland (n = 4939) enrolled into multicenter clinical trial AIEOP-BFM ALL 2000 between July 1999 and June 2009. Patients enrolled into study AIEOP-BFM ALL 2009 - which was initiated subsequent to AIEP-BFM ALL 2000 - who were reported with a cancer prone syndrome or chromosomal abnormality were additionally included in this study to increase conclusiveness of observations. RESULTS: A total of 233 patients with at least one reported condition could be identified. The following conditions were reported in more than one patient: Gilbert's disease (n = 13), neurofibromatosis type I (n = 8), ataxia telangiectasia (n = 8), thalassemia (n = 7), Nijmegen Breakage syndrome (n = 6), cystic fibrosis (n = 4), glucose-6-phosphate dehydrogenase deficiency (n = 4), Noonan syndrome (n = 2), Klinefelter syndrome (n = 2), alpha-1-antitrypsin deficiency (n = 2), primary ciliary dyskinesia (n = 2). Especially those syndromes with a known cancer predisposition (NF type I, Ataxia telangiectasia, Nijmegen Breakage syndrome etc.) were associated with certain general and ALL-related characteristics, high therapy-related toxicity and reduced survival. CONCLUSION: The spectrum of underlying diseases within ALL patients is dispersed. A small number of ALL patients are reported with cancer predisposition syndromes at initial diagnosis which are associated with high rates of therapy-related toxicity and a markedly reduced chance of survival. The true prevalence of these conditions within the ALL population remains unknown due to inapparent clinical presentation. A targeted clinical and/or genetic examination for certain diagnoses like NF type I, Ataxia telangiectasia or Nijmegen Breakage syndrome could identify patients who benefit from adjustment of antileukemic therapy or intensification of supportive care.

Prognostic value of rare IKZF1 deletion in childhood B-cell precursor acute lymphoblastic leukemia: an international collaborative study.

Deletions in IKZF1 are found in ~15% of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL). There is strong evidence for the poor prognosis of IKZF1 deletions affecting exons 4-7 and exons 1-8, but evidence for the remaining 33% of cases harboring other variants of IKZF1 deletions is lacking. In an international multicenter study we analyzed the prognostic value of these rare variants in a case-control design. Each IKZF1-deleted case was matched to three IKZF1 wild-type controls based on cytogenetic subtype, treatment protocol, risk stratification arm, white blood cell count and age. Hazard ratios for the prognostic impact of rare IKZF1 deletions on event-free survival were calculated by matched pair Cox regression. Matched pair analysis for all 134 cases with rare IKZF1 deletions together revealed a poor prognosis (P<0.001) that was evident in each risk stratification arm. Rare variant types with the most unfavorable event-free survival were DEL 2-7 (P=0.03), DEL 2-8 (P=0.002) and DEL-Other (P<0.001). The prognosis of each type of rare variant was equal or worse compared with the well-known major DEL 4-7 and DEL 1-8 IKZF1 deletion variants. We therefore conclude that all variants of rare IKZF1 deletions are associated with an unfavorable prognosis in pediatric BCP-ALL.

Transplantation of allogeneic CD34-selected stem cells after fludarabine-based conditioning regimen for children with mucopolysaccharidosis 1H (M. Hurler).

Hurler syndrome (MPS1H) is a progressive inborn error of mucopolysaccharide metabolism leading to premature death. Allogeneic hematopoietic cell transplantation (HCT) can achieve stabilization and improve long-term survival. However, large studies have shown that preparative regimen-related toxicity (RRT) and graft failure rates have been relatively high. We transplanted five Hurler children with a fludarabine-based conditioning regimen, consisting of fludarabine/busulphan/ATG for matched family donor (MFD), with the addition of melphalan for mismatched family donor and matched unrelated donor (MUD) transplantations. Median age at HCT was 27 months (range 10-36). The source of stem cells was bone marrow in one MFD and CD34-selected PBSC in four patients. Median CD34+ cell dose was 25 x 10(6)/kg (range 11.5-54). No RRT > grade II was observed. All patients are surviving at a median of 32 months (range 14-41) and show sustained donor engraftment with 3/5 having full donor chimerism, and 2/5 mixed chimerism (> 85%). We conclude that this regimen is feasible and has low toxicity in Hurler children. In combination with high doses of CD34+ selected cells (> 10 x 10(6)/kg) and donor lymphocyte infusions, stable engraftment could be achieved in unrelated and mismatched related transplantations.

Topoisomerase II inhibitors induce DNA double-strand breaks at a specific site within the AML1 locus.

Treatment-related acute myeloid leukemia (t-AML) following successful therapy of a primary malignancy has been recognized with increasing frequency among cancer survivors over the past several years. Many of these t-AML cases are associated with the use of intensive chemotherapy regimens that employ one or more agents which target eukaryotic topoisomerase II (topo II), and demonstrate non-random chromosomal translocations involving either the MLL (ALL-1, HRX) gene at 11q23 or the AML1 gene at 21q22. Although many investigators have speculated that these translocations are induced by the therapeutic use of topo II inhibitors, the molecular sequence of events by which topo II inhibitors might induce a chromosomal translocation are not well understood. We describe here the reproducible induction of highly specific, double-strand DNA cleavage at a specific site within the AML1 locus by topo II inhibitors. This DNA cleavage, which maps to a region of the AML1 locus frequently disrupted by chromosomal translocations, can be induced in several cell lines, with multiple different topo II inhibitors, indicating that this phenomenon is not restricted to a specific cell type or specific topo II inhibitor. It is conceivable that site-specific double-strand DNA cleavage within the AML1 locus induced by topo II inhibitors represents the initial molecular event leading to a chromosomal translocation and t-AML.

Association of initial response to prednisone treatment in childhood acute lymphoblastic leukaemia and polymorphisms within the tumour necrosis factor and the interleukin-10 genes.

Plasma levels of TNF and IL-10 have been associated with therapy outcome in haematological malignancies and are influenced by genetic variation due to germline polymorphisms within the TNF and IL-10 genes. Different TNF and IL-10 genetic polymorphisms might therefore also correlate with clinical outcome in childhood acute lymphoblastic leukaemia (ALL). We analysed the association of TNF and IL-10 polymorphisms with response to initial treatment and risk of relapse in 135 children with ALL, treated according to Berlin-Frankfurt-Münster (BFM) protocols. Our data showed a protective effect from prednisone poor response in patients with the IL-10 G/G genotype, whereas no association of the risk of relapse and IL-10 genotype was found. In the total study group, subjects expressing the TNF2 allele neither showed a statistically significant general association with prednisone response nor with risk of relapse compared to subjects homozygous for the TNF1 allele. Nevertheless, we did find a higher risk of relapse in poor prednisone responders expressing the TNF2 allele compared to poor prednisone responders not expressing the TNF2 allele. We conclude that IL-10 genotype might influence prednisone response in patients with childhood ALL, whereas TNF genotype seems to influence the risk of relapse in high risk ALL patients.

Infant acute lymphoblastic leukemia - combined cytogenetic, immunophenotypical and molecular analysis of 77 cases.

We used karyotyping, fluorescence in situ hybridization (FISH), Southern blotting, and RT-PCR in order to analyze prospectively 77 infants (less than 1 year of age) with acute lymphoblastic leukemia for the occurrence of 11q23/MLL rearrangements and/or other cytogenetic abnormalities. Out of the 69 informative samples we found an 11q23/MLL rearrangement in 42 cases (61%). Regarding only pro-B ALL cases, the incidence of 11q23/MLL rearranged cases, however, reached more than 90% The infants were treated within the therapy studies ALL-BFM90, ALL-BFM95 and CoALL-05-92. For patients with an adequate follow-up of 4 years the event-free survival of the 11q23/MLL-positive and 11q23/MLL-negative group was 0.2 or 0.64, respectively (P = 0.024). The monoclonal antibody 7.1. (moab 7.1) does not react with normal hematopoetic precursors or mature blood cells but was shown to specifically react with leukemic cells bearing a rearrangement of chromosome 11q23 or the MLL gene, respectively. We, therefore, specifically addressed the question whether the reactivity of moab 7.1, as determined by flow cytometry, may substitute for molecular testing of an 11q23/MLL rearrangement in this cohort of infant ALLs. Reactivity of moab 7.1 indicated a 11q23/MLL rearrangement with a specificity of 100%. However, five of the 11q23/MLL-positive cases did not react with moab 7.1 indicating a sensitivity of 84% only. Three of these five moab 7.1-negative but 11q23/MLL-positive cases could be identified by their unique expression pattern of CD65s and/or CD15. Thus, 95% of all 11q23/MLL-positive ALL cases in infancy may be identified by flow cytometry based on their expression of CD15, CD65s and/or moab 7.1.